Project 2 (Pipkin) Activation of naive CD8 T cells during intracellular infections rapidly induces chromatin remodeling and transcriptional reprogramming that results in the differentiation of memory (TMEM) CTLs that provide long-term immunity. We discovered that the transcription factor (TF) Runx3 instigates chromatin accessibility of TMEM- associated cis-regulatory regions in naive cells during initial T cell receptor (TCR) stimulation, and is essential for the differentiation of both circulating and tissue resident TMEM CTLs. Runx3 activates transcriptional circuits that establishes nascent CTLs, represses alternative cell fates and prevents terminal CTL differentiation. Using mass spectrometry we identified all subunits of the NuRD/HDAC complex in association with Runx3. In addition, we conducted a pooled, RNA interference (RNAi) -mediated, loss-of-function screen in CD8 T cells responding to viral infection that targeted all mammalian chromatin regulator factors (CRFs, 312 genes). This revealed that deficiency in multiple individual NuRD/HDAC complex subunits impaired memory precursor CTL differentiation, similar to Runx3 loss-of-function. In addition, multiple subunits of the BAF-family of nucleosome remodelers and its collaborator Chd7, which is mutated in a human immunodeficiency, were essential for initiating very early aspects of CTL differentiation and driving terminal differentiation. To gain insight into early aspects of CTL development, we used a single cell RNA-seq (scRNA-seq) approach and performed computational trajectory analyses which identified common, and then branching developmental pathways from naive CD8s that lead toward effector and memory cell fates. In part, we confirmed these pathways operationally using Blimp1-YFP reporter alleles and adoptive transfer experiments. In the current application, we propose to build on these results to elucidate how CRFs and TFs reprogram chromatin structure during naive cell activation and early establishment of effector and memory-like developmental paths. Specifically, we will define how Runx3 and NuRD/HDAC complexes remodel chromatin structure to establish initial TMEM transcriptional programs (Aim 1). We will elucidate how hierarchical functions of Runx3, Ets1, Blimp1 and Bcl6 regulate the divergence of Blimp1hi and Blimp1lo effector- and memory-like developmental paths, and use an in vivo conditional RNAi approach in naive CD8s to screen all T cell-expressed TFs (1,751 genes) to identify their roles in this process during viral infection (Aim 2). Finally, we will integrate these analyses with how BAF and Chd7 remodelers govern nucleosome organization in cis-regulatory regions that control transcriptional reprograming during early CTL differentiation (Aim 3). These studies integrate synergistically with analogous approaches addressing the roles of CRFs and TFs in TFH differentiation and function (Crotty, Project 1), and how they function at later times to maintain the differentiation and function of specific CD4 and CD8 TMEM cell subsets that persist following infections, and that infiltrate tumors (Goldrath, Project 3). 1